EP0519976B1 - Neutrophile stimulierende peptide - Google Patents

Neutrophile stimulierende peptide Download PDF

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Publication number
EP0519976B1
EP0519976B1 EP91905955A EP91905955A EP0519976B1 EP 0519976 B1 EP0519976 B1 EP 0519976B1 EP 91905955 A EP91905955 A EP 91905955A EP 91905955 A EP91905955 A EP 91905955A EP 0519976 B1 EP0519976 B1 EP 0519976B1
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EP
European Patent Office
Prior art keywords
peptide
peptides
amino acid
acid sequence
amino acids
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Expired - Lifetime
Application number
EP91905955A
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English (en)
French (fr)
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EP0519976A1 (de
EP0519976A4 (en
Inventor
Deborah Anne Rathjen
Antonio Ferrante
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Teva Pharmaceuticals Australia Pty Ltd
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Peptide Technology Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to peptides having neutrophil stimulating activity, and to use of these peptides as therapeutic agents.
  • Tumour necrosis factor was first identified as a factor found in the serum of Bacillus Calmette-Guerin treated mice which caused haemorraghic regression of certain transplanted tumours and had cytolytic activity on several transformed cell lines in vitro (Carswell et al, PNAS 72 , 3666 - 3670; Helson et al, 1975, Nature 258 , 731-732).
  • TNF a product of activated macrophages, has subsequently been shown to be a primary mediator in the pathology of endotoxic shock (Tracey et al 1986, Science 234 , 470-474).
  • TNF also has a central role in host defenses against viral, bacterial and parasitic pathogens.
  • TNF TNF-binding protein
  • monocyte/macrophages TNF-binding protein
  • lymphocytes TNF-binding protein
  • TNF neutrophil activation protein
  • TNF stimulates enhanced phagocytosis (Shalaby et al 1985, J.Immunol., 135 , 2069-2073), enhanced production of superoxide anions (Teujiimoto et al, 1986, Biochem. Biophys. Res. Commun., 137 , 1094-1100), release of lysozyme and hydrogen peroxide and causes neutrophil degranulation (Klebanoff et al, 1986, J.Immunol., 136 , 4220-4225).
  • Neutrophils also show enhanced microbicidal and tumouricidal activity when stimulated by TNF (Shalaby et al, 1985, J.Immunol., 135 , 2069-2073; Djeu et al, 1986, J.Immunol., 137 , 2980-2984; Blanchard et al, 1989, J.Leuk. Biol., 45 , 538-545). It has been hypothesized that the cytostatic effect of TNF is mediated by high local concentrations of hydrogen peroxide produced by neutrophils (Shau 1986, J.Immunol., 141 , 234-240).
  • TNF pretreatment enhances the response of neutrophils to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (F-met-leu-phe) and phorbol myristate acetate through specific receptors (Ferrante et al 1988, Int. Arch. Allergy Appl. Immunol., 86 , 82-91).
  • Neutrophils accumulate at sites of inflammation, caused in part by the increased expression of complement receptors by TNF (Berger et al 1988, Blood 71 , 151-158). Further TNF causes neutrophil emigration into skin (Cybuisky et al 1988, J. Immunol. 140 , 3144-3149).
  • Neutrophil function is known to be depressed in a number of viral, bacterial and parasitic infections (Abramson and Mills, 1988, Rev. Infect. Dis., 10 , 326-341; Ferrante et al, 1989, Immunol. Letts., 22 , 301-6).
  • Depressed neutrophil function has, for example, been described in Acquired Immune Deficiency Syndrome (Thorsen et al, 1989, AIDS, 3, 651-653; Ellis et al, 1988, J. Infect. Dis., 158 , 1268-1276; Murphy et al, 1988, J. Infect. Dis., 158 , 627-630).
  • TNF which appears to play an important role in neutrophil activation both in vitro and in vivo as described above, given exogenously has the potential to overcome these neutrophil defects.
  • the administration of TNF or indeed overproduction of TNF is, however, associated with severe side effects and the manifestation of pathology such as thrombocytopaenia, lymphocytopaenia, hepatotoxicity, renal impairment and hypertension.
  • Socher et al disclose a peptide having an amino acid sequence corresponding to the 65-79 fragment of TNF.
  • the present inventors have identified novel peptides derived from the primary amino acid sequence of human TNF which stimulate neutrophil activity. These peptides have indicated that the region of amino acids 54 to 94 of human TNF has previously undiscovered neutrophil stimulating activity. This observation has important clinical applications as treatment with such peptides would be expected to restore depressed or aberrant neutrophil activity, but would not be expected to cause the severe side effects associated with the therapeutic use of the whole TNF molecule.
  • the present invention consists in a peptide having an amino acid sequence substantially corresponding to amino acids 54 to 94 of Figure 1 or a part thereof, the peptide being characterized in that the peptide is capable of eliciting superoxide production by neutrophils and of priming neutrophils for an enhanced respiratory burst following treatment with N-formyl-L-methionyl-L-leucyl-L-phenylalanine.
  • the peptide has an amino acid sequence corresponding to amino acids 54 to 94 of Figure 1.
  • the peptide has an amino acid sequence corresponding to amino acids 63 to 83 of Figure 1.
  • the peptide has an amino acid sequence corresponding to amino acids 54 to 68 of Figure 1.
  • the peptide has an amino acid sequence corresponding to amino acids 73 to 94 of Figure 1.
  • the peptide has amino acid sequence corresponding to amino acids 70 to 80 of Figure 1.
  • the present inventors have demonstrated that the region of human TNF from amino acid 54 to amino acid 94 plays an important functional role in the stimulation of neutrophils. Further, the present inventors have produced 4 peptides namely peptides 304, 308, 309 and 395 (as referred to herein) which have neutrophil stimulating activity.
  • the present invention consists in a method of treating a subject having depressed neutrophil function, the method comprising administering to the subject a therapeutic amount of the peptide of the first aspect of the present invention.
  • the subject is suffering from acquired immune deficiency syndrome.
  • Peptide 308 through slective effects on neutrophil degranulation may be administered to individuals suffering from inflammatory syndromes e.g. rheumatoid arthritis, adult respiratory distress syndrome.
  • inflammatory syndromes e.g. rheumatoid arthritis, adult respiratory distress syndrome.
  • the carboxy terminal amino acid is attached to the solid support by a DCC/DMAP-mediated symmetrical-anhydride esterification.
  • Peptide 304 Peptide is cleaved from the resin with 95% TFA and 5% phenol (5 h) and purified on reverse phase C4 column. (Buffer A - 0.1% aqueous TFA, Buffer B - 80% ACN 20% A).
  • Peptide 308 Peptide is cleaved from the resin with 95% TFA and 5% water (1.5 h) and purified on reverse phase C4 column. (Buffer A - 0.1% aqueous TFA, Buffer B - 80% ACN 20% A).
  • Peptide 309 Peptide is cleaved from the resin with 95% TFA and 5% thioanisole and purified on reverse phase C4 column. (Buffer A - 0.1% aqueous TFA, Buffer B - 80% ACN 20% A).
  • Chemiluminesence assay .
  • This assay examined the effect of TNF peptides on priming for a neutrophil F-met-leu-phe response as described by Ferrante et al, 1988, (Int. Arch. Allergy Appl. Immunol, 86, 82-91). Purified human neutrophils were pretreated with peptide for 20 minutes before the addition of f-met-leu-phe. The lucigenin dependent chemiluminescence response, which reflects superoxide production, was then measured. The results obtained are set out in Table 2 and are expressed as mV of lucigenin dependent chemiluminescence and represent the maximal cell activity attained.
  • TNF and peptide 304, 308 and 309 are important properties by which cells reach infection sites. Their accumulation at these sites is also dependent on the capacity of inflammatory mediators to inhibit their migration out of the sites.
  • the present inventors have examined TNF and peptide 304, 308 and 309 for their effect on the migration of neutrophils.
  • neutrophils were pre-treated with the peptide or TNF and then examined their ability to migrate out of wells in agarose as described by Ferrante et al, 1988, (Arch. Allergy Appl. Immunol. 86 :82-91). The results are shown in Table 7. The results show that TNF was only partially migration inhibitory at 100 units/10 6 cells. Both peptides 308 and 309 were potent migration inhibitors, however, peptide 304 was found to be chemokinetic (it stimulated cell migration).
  • TNF and peptides 304, 308 and 309 were examined using the following method: 3ml of molten 2% agarose was mixed with 3ml of 2x concentrated medium 199 containing foetal calf serum (10%) and poured into Petri dishes. Sets of 3 wells of 2.5mm diameter, each 3mm apart, were cut in the agarose. 5 ⁇ l of neutrophils (2 x 10 5 cells) were added to the inner well, with chemotactic agent or control medium added to the outer wells. Migration at various time intervals was then measured. The results of these experiments are shown in Table 8.
  • Vitamin B 12 binding protein was measured using 57 Co-vitamin B 12 . This assay is based on the principle that the binding protein binds to the 57 Co-Vitamin B 12 and as a result the radioactive vitamin B 12 does not bind to charcoal.
  • TNF ⁇ peptides 304, 308 and 309 were found to prime human neutrophils for the respiratory burst associated with f-met-leu-phe treatment, i.e. in a manner analogous to that of TNF ⁇ . Together these peptides comprise the primary amino acids sequence region of amino acids 54 to 94 of human TNF ⁇ . Peptide 308 is a particularly potent primer of neutrophils in this assay.
  • peptide 323 which has a sequence which corresponds to amino acids 79 to 89 of human TNF was not found to be capable of priming neutrophils for the respiratory burst associated with f-met-leu-phe treatment.
  • the reason for the lack of neutrophil stimulating activity of this peptide has not as yet been ellucidated, however, one hypothesis for the lack of activity of this peptide may be that peptide 323 does not include the amino acid residues which bind to the TNF receptor on the neutrophils.
  • Peptides 308 and 309 have also been found to be potent inhibitors of neutrophil migration whilst peptide 304 has been found to be chemokinetic. Peptide 308 has also been found to be strongly chemotactic.
  • TNF peptides 304 and 308 were tested for their effects on degranulation of neutrophils (Table 9) for their effects.
  • peptide 308 decreased the release of the contents of both the specific and the azurophilic granules as measured by the release of Vitamin B 12 binding protein and ⁇ -glucuronidase release respectively.
  • This effect of peptide 308 was still apparent following stimulation with fMLP.
  • peptide 304 had no effect on neutrophil degranulation in the absence of fMLP.
  • fMLP peptide 304 enhanced release from specific granules but not azurophilic granules.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
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  • Toxicology (AREA)
  • Diabetes (AREA)
  • Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Hematology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Claims (12)

  1. Peptid mit einer Aminosäuresequenz, die den Aminosäuren 54 bis 94 von Figur 1 entspricht, oder ein Teil davon, wobei das Peptid Neutrophile zur Superoxidproduktion und zu einer erhöhten Stoffwechselaktivierung ("respiratory burst") im Anschluß an eine Behandlung mit N-Formyl-L-methionyl-L-leucyl-L-phenylalanin anregen kann.
  2. Peptid nach Anspruch 1, wobei das Peptid eine Aminosäuresequenz aufweist, die den Aminosäuren 54 bis 94 in Figur 1 entspricht.
  3. Peptid nach Anspruch 1, wobei das Peptid eine Aminosäuresequenz aufweist, die den Aminosäuren 54 bis 68 in Figur 1 entspricht.
  4. Peptid nach Anspruch 1, wobei das Peptid eine Aminosäuresequenz aufweist, die den Aminosäuren 73 bis 94 in Figur 1 entspricht.
  5. Peptid nach Anspruch 1, wobei das Peptid eine Aminosäuresequenz aufweist, die den Aminosäuren 70 bis 80 in Figur 1 entspricht.
  6. Verbindung mit einer dreidimensionalen Struktur, die der dreidimensionalen Struktur des Peptids gemäß einem der Ansprüche 1 bis 5 entspricht, wobei die Verbindung Neutrophile zu einer Stoffwechselaktivierung ("respiratory burst") im Anschluß an eine Behandlung mit N-Formyl-L-methionyl-L-leucyl-L-phenylalanin anregen kann.
  7. Arzneimittel, umfassend ein Peptid nach einem der Ansprüche 1 bis 5 und einen pharmazeutisch verträglichen Träger oder Verdünnungsmittel.
  8. Peptid nach einem der Ansprüche 1 bis 5 oder eine Verbindung nach Anspruch 6 zur Verwendung in der Medizin.
  9. Verwendung eines Peptids nach einem der Ansprüche 1 bis 5 oder eine Verbindung nach Anspruch 6 für die Herstellung eines Mittels zur Behandlung oder Prophylaxe von Krankheiten, die durch eine verminderte neutrophile Funktion gekennzeichnet sind.
  10. Verwendung nach Anspruch 9, wobei das Individuum entweder am erworbenen Immunschwächesyndrom oder an Krebs leidet.
  11. Verwendung eines Peptides nach Anspruch 3 für die Herstellung eines Mittels zur Behandlung eines Individuums, das an einem Entzündungssyndrom leidet.
  12. Verwendung nach Anspruch 11, wobei das Entzündungssyndrom, an dem das Individuum leidet, rheumatoide Arthritis oder die akute respiratorische Insuffizienz ist.
EP91905955A 1990-03-12 1991-03-12 Neutrophile stimulierende peptide Expired - Lifetime EP0519976B1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AUPJ906590 1990-03-12
AU9065/90 1990-03-12
PCT/AU1991/000086 WO1991013908A1 (en) 1990-03-12 1991-03-12 Neutrophil stimulating peptides

Publications (3)

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EP0519976A1 EP0519976A1 (de) 1992-12-30
EP0519976A4 EP0519976A4 (en) 1993-06-09
EP0519976B1 true EP0519976B1 (de) 1997-02-26

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EP91905955A Expired - Lifetime EP0519976B1 (de) 1990-03-12 1991-03-12 Neutrophile stimulierende peptide

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EP (1) EP0519976B1 (de)
JP (1) JP3241041B2 (de)
AU (1) AU642487B2 (de)
CA (1) CA2078000C (de)
DE (1) DE69124795T2 (de)
DK (1) DK0519976T3 (de)
SG (1) SG46398A1 (de)
WO (1) WO1991013908A1 (de)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5587457A (en) * 1990-03-12 1996-12-24 Peptide Technology Limited Neutrophil stimulating peptides
US6375928B1 (en) * 1990-03-12 2002-04-23 Peptech Limited Neutrophil stimulating peptides
WO1991014702A1 (en) * 1990-03-19 1991-10-03 Peptide Technology Ltd. Anti-tumour peptides
WO1997048725A1 (en) * 1996-06-21 1997-12-24 Peptech Limited Novel peptides for prevention and treatment of infection
US7851486B2 (en) 2002-10-17 2010-12-14 Decode Genetics Ehf. Susceptibility gene for myocardial infarction, stroke, and PAOD; methods of treatment

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GR851626B (de) * 1984-07-05 1985-11-26 Genentech Inc
EP0486526B2 (de) * 1989-08-07 2001-03-07 Peptech Limited Bindeligande für tumornekrosisfaktor
WO1991014702A1 (en) * 1990-03-19 1991-10-03 Peptide Technology Ltd. Anti-tumour peptides
WO1991017180A1 (en) * 1990-04-27 1991-11-14 Peptide Technology Ltd Peptides derived from human tumour necrosis factor alpha and useful against intracellular malarial parasites

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Proc. Natl. Acad. Sci USA, Vol. 84, pp. 8829-8833, Dec. 1987, S.H. SOCHER et al. *

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DE69124795D1 (de) 1997-04-03
WO1991013908A1 (en) 1991-09-19
DK0519976T3 (da) 1997-04-01
EP0519976A1 (de) 1992-12-30
AU642487B2 (en) 1993-10-21
DE69124795T2 (de) 1997-06-26
CA2078000A1 (en) 1991-09-13
EP0519976A4 (en) 1993-06-09
SG46398A1 (en) 1998-02-20
CA2078000C (en) 2001-10-02
JPH05506850A (ja) 1993-10-07
JP3241041B2 (ja) 2001-12-25
AU7476291A (en) 1991-10-10

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